Within-person reproducibility of proteoforms related to inflammation and renal dysfunction

Protein biomarkers and microheterogeneity have attracted increasing attention in epidemiological and clinical research. Knowledge of within-person reproducibility over time is paramount to determine whether a single measurement accurately reflects an individual’s long-term exposure. Yet, research investigating within-person reproducibility for proteoforms is limited. We investigated the reproducibility of the inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), and calprotectin (S100A8/9), and the renal function marker cystatin C (CnC) using a novel immuno-MALDI-TOF MS assay. Reproducibility, expressed as intraclass correlation coefficient (ICC), was calculated for 16 proteoforms using plasma samples of the Western Norway B Vitamin Intervention Trial (WENBIT) cohort collected 1–3 y apart from 295 stable angina pectoris (SAP) patients and 16 weeks apart from 38 subjects of the Intervention with Omega Fatty Acids in High-risk Patients with Hypertriglyceridemic Waist (OMEGA) trial with abdominal obesity but no other documented co-morbidities. ICCs for inflammatory markers were lower in WENBIT (CRP: 0.51, SAAt: 0.38, S100At: 0.31) compared to OMEGA subjects (CRP: 0.71, SAAt: 0.73, S100At: 0.48), while comparable for CnCt (WENBIT: 0.69, OMEGA: 0.67). Excluding SAP patients with elevated inflammation (CRP > 10 µg/ml) increased the ICC of SAAt to 0.55. Reduction of the time interval from 3 to 1 y in WENBIT group increased ICCs for all proteoforms. With a few exceptions ICCs did not differ between proteoforms of the same biomarker. ICCs were highest in OMEGA subjects with fair-to-good reproducibility for all markers. Reproducibility of SAA and S100A8/9 proteoforms in the WENBIT cohort was related to inflammation. This work will inform future clinical and epidemiological research which relies on single time point biomarker assessment to investigate inflammation and renal function.


Scientific Reports
| (2022) 12:7426 | https://doi.org/10.1038/s41598-022-11520-1 www.nature.com/scientificreports/ We investigated the within-person reproducibility of the inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), and calprotectin (S100A/9), and the renal function marker cystatin C (CnC) using a novel immuno-MALDI-TOF MS assay 13 . While ICCs have been reported earlier for the total protein concentrations of these markers, reproducibility of the 16 different proteoforms is unknown. ICCs of biomarkers may vary between cohorts (lifestyle, gender, clinical status, etc.) and study designs (size, duration, number of sampling intervals). Thus, we choose samples from two quite different studies, representing clinical patients and subjects with abdominal obesity but no other documented co-morbidities, to illustrate the potential for ICC variability of the four investigated biomarkers, which we think is especially important when comparing ICCs of the investigated protein biomarkers to those reported in the literature.

Study populations and sample collection. Within-person reproducibility over time was investigated
in two different cohorts, Western Norway B Vitamin Intervention Trial (WENBIT) and Intervention With Omega Fatty Acids in High-risk Patients with Hypertriglyceridemic Waist (OMEGA), consisting of 295 stable angina pectoris (SAP) patients and 38 subjects with abdominal obesity but on other documented co-morbidities, respectively. The clinical characteristics of the participants have been summarized in Table 1 for both cohorts.
Patients of the WENBIT cohort were randomly selected from the placebo control group of this study 14 , collected over a period of 3 years, and who suffered from stable angina pectoris (SAP) and had undergone coronary angiography for suspected coronary artery disease. All WENBIT participants provided written informed consent. The study protocol was in accordance with the principles of the Declaration of Helsinki and was approved by the Regional Committee for Medical and Health Research Ethics, the Norwegian Medicines Agency, and the Data Inspectorate. The ClinicalTrials.gov identifier was NCT00354081.
Blood samples of the OMEGA trial 15 were collected over a period of 16 weeks. OMEGA subjects had participated in a crossover intervention study investigating the effects of omega-3 and omega-6 oil supplementation. Briefly, the study included volunteers who had increased waist circumference (≥ 94 cm in men, ≥ 80 cm in women) and were physical inactive (< 2 h vigorous/active exercise training per week). Samples taken at baseline (week 0) and after a wash-out phase at week 16 (7 weeks of the first intervention period plus 9 weeks wash-out phase) were utilized for the purpose of the present study. The OMEGA study was conducted according to the guidelines in the Declaration of Helsinki, and was approved by the Regional Committee for Medical and Health Research Ethics (2014/2336/REK South-East). The written informed consent was obtained from each participant before study.
All samples investigated were EDTA plasma samples, stored at -80 °C within 30 min after collection.
Laboratory analyses. Samples were analyzed by a novel immuno-MALDI-TOF MS assay described previously 13 . Briefly, 20 µL EDTA plasma were spiked with 20 µL internal standards of polyhistidine-tagged recombinant proteins and incubated with antibody-immobilized paramagnetic beads. After intensive washing, proteins were eluted from the beads and analyzed by MALDI-TOF MS. Samples were processed in 96-microtiter plates using a Hamilton MircolabStar (Bonaduz, Switzerland) and CyBi-Disk robot from Analytik Jena (Jena, Germany).
Nomenclature of proteoforms. SAAt, S100A8/9t and CnCt represented the total concentrations of the corresponding proteins. N-terminally truncated SAA, S100A8/9 and CnC were labelled with a "d" and the one-letter code(s) of the missing amino acids. SAA proteoforms were abbreviated according to the isoforms expressed by the SAA1 or SAA2 gene. Monomers of S100A8/9 were denoted as S100A8 and S100A9. S100A9tr was short for the shortest truncation of S100A9 which missed 5 amino acids. The native and the hydroxylated forms of CnC were abbreviated as CnCn and CnCo, respectively 16 .

Statistical analyses.
Age, body mass index (BMI), waist circumference and estimated glomerular filtration rate (eGFR) of the participants in both cohorts were indicated as arithmetic mean (SD). Protein and proteoform www.nature.com/scientificreports/ concentrations were presented as geometric means with 95% CIs. Proteoform concentrations were determined either as absolute levels or as values relative to the total concentration of the biomarker. Deviation of geometric mean concentrations between time points were determined by Student paired t-test. Correlation of biomarker concentrations between baseline (BL) and end of follow-up (END) were investigated by Spearman rank test. Within-person reproducibility was expressed as ICC and calculated using ln-transformed values and an ICC (1,1) model 17 . ICCs were classified according to Rosner as poor (< 0.4), fair-to-good (0.4-0.75), and excellent (≥ 0.75) 2 . Within-and between-person CVs were determined by calculating the square root of the variance components from the random-effect mixed model and were classified as high (> 100%), moderate (50-100%) and low (< 50%). The program R version 3.5.3 was used for statistical analyses, and the packages "DescTools", "stat" and "ICC" (ICCest function) were used for geometric mean (95% CIs), Spearman rank test, Student t-test and ICC calculation, respectively.

Results
Total concentrations across time. The total concentrations of the four protein biomarkers determined in the WENBIT and OMEGA cohorts are presented in Fig. 1. Levels of CRP and CnCt were comparable in both cohorts, while concentrations of SAAt and S100At were higher in WENBIT than OMEGA. Levels of CRP did not change during the follow up periods in WENBIT and OMEGA. Total plasma concentrations of SAA, S100A and CnC were stable in OMEGA, but changed in WENBIT. Plasma levels of S100At and CnCt increased significantly during the 3 years of follow up in WENBIT.

Within-and between-person variability of biomarkers and proteoforms. The geometric mean
(for all time points) for total protein and proteoform, Spearman correlation (BL vs END), within-and betweenperson coefficient of variation (CV), and ICC (95% CI) are shown for WENBIT ( Table 2) and OMEGA (Table 3) participants.
In accordance with the total biomarker concentrations, plasma levels of S100A8/9 and most CnC proteoforms increased significantly over time in the WENBIT group (Table 2). Within-person CVs were high for CRP and S100A8/9, moderate for SAA, and low for CnC. Similar CVs were obtained for between-person variation, with the exception of S100A8/9, which demonstrated moderate variation. Samples were collected at 3 visits over 3y from 295 SAP patients enrolled in WENBIT and at 2 visits over 16 weeks from 38 participants in OMEGA. The concentrations were presented as geometric mean with 95% CIs, and the difference between concentrations for any two visits, investigated by t-test, were indicated in the order of BL versus 1Y, BL versus END, and 1Y versus END for WENBIT, and BL versus END for OMEGA. NS Not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. www.nature.com/scientificreports/ For OMEGA, plasma levels of proteoforms did not differ significantly between baseline and end (Table 3). Within-person CVs were moderate for CRP and SAA, and low for CnC. Variance differed between S100A8/9 proteoforms and ranged from moderate (S100A9, S100A9dm) to low (S100A8, S100A9tr). Between-person CVs of S100A8/9 and CnC were similar to their within-person variation, whereas between-person CVs for CRP and SAA were higher.

Scientific Reports
Within-person reproducibility of biomarkers and proteoforms. In WENBIT (Table 2), the ICCs of CRP and CnC were highest among the protein biomarkers, and ranged between 0.41 and 0.73, while ICCs of SAA and S100A8/9 were lower, ranging from 0.28 to 0.44. ICCs were slightly higher than Spearman's Rho for CRP, S100A8/9, and CnC, and lower for SAA. Reproducibility of CRP, SAA and CnC in OMEGA (Table 3) were similar and ICCs ranged from 0.58 to 0.77. ICCs of S100A8/9 was lower and varied from 0.35 to 0.54. ICCs were higher than Spearman's Rho for CRP and CnC, and comparable for SAA and S100A8/9. However, due to Table 2. Concentrations and within-person reproducibility of biomarkers in EDTA plasma samples from WENBIT. Samples collected at 3 time points over 3y apart from 295 SAP patients enrolled in the Western Norway B Vitamin Intervention Trial. Within-and between-person CVs were determined by taking the square root of the within-and between-person variance components from random-effects mixed model on the ln-transformed values. a BL Baseline, END End of follow-up. b Significant differences in biomarker geometric mean were evaluated by t-test. c Values were obtained from the data at BL and END. www.nature.com/scientificreports/ the low number of subjects in the OMEGA trial, 95% CIs for ICCs were 2-3 times larger than those observed in WENBIT. ICCs of total protein and proteoform concentrations were compared between cohorts and illustrated as radar plots (Fig. 2). In WENBIT, ICCs ranged from fair-to-good for CRP, and poor for SAA and S100A8/9. In contrast, fair-to-good reproducibility was observed for most proteoforms in OMEGA, with ICCs for CRP and SAA close to excellent. SAA1.1 in the OMEGA cohort was the only proteoform demonstrating excellent reproducibility. ICCs of CnC were fair-to-good and showed comparable values in both study groups. Notably, differences in ICCs between proteoforms of the same biomarker were generally small with exception of a few low-abundance proteoforms.
Additional sub-group analyses were performed in the WENBIT cohort. The impact of acute inflammation on ICCs was investigated by excluding 29 patients with CRP > 10 µg/ml (Fig. 3A). Removal of those with elevated CRP (reflective of increased acute systemic inflammation) improved the ICCs of all inflammatory markers. While the increase was marginal for CRP and S100A8/9, ICCs of SAA proteoforms increased markedly and changed performance from poor to fair-to-good. Furthermore, ICCs were calculated for the different time intervals between baseline and 1 or 3 years (Fig. 3B). While ICCs for CRP and SAA were similar for both time intervals, reproducibility for S100A8/9 and CnC proteoforms were highest after 1 year follow up. Notably, the reproducibility of CnCn and CnCo were excellent at 1 year, fair-to-good at 3 years.

Variability of proteoform distributions across observation period. The variability of proteoform
distributions was investigated in both cohorts by comparing the geometric means of relative proteoform concentrations (Fig. 4) and the within-and between-person variances based on both absolute and relative levels (Supplemental Fig. 1). Relative levels of SAA, S100A8/9 and CnC proteoforms were stable over time in both cohorts. However, weak but significant differences (p < 0.05) were observed for S100A8/9 and CnC in WENBIT. In addition, within-and between-person variances of relative values were generally lower than for absolute proteoform concentrations (Supplemental Fig. 1), and ranged between 5-52% and 3-82% in WENBIT and OMEGA, respectively. Lowest variation was observed for the proteoforms of CnC with an average CV of 10%. Table 3. Concentrations and within-person reproducibility of biomarkers in EDTA plasma samples from OMEGA. Samples collected at 2 time points over 16 weeks apart from 38 subjects in the OMEGA Trial. Within-and between-person CVs were determined by taking the square root of the within-and betweenperson variance components from random-effects mixed model on the ln-transformed scale. a BL Baseline, END End of follow-up. b Significant differences in biomarker geometric mean were evaluated by t-test.

Discussion
Biomarker levels and within-person reproducibility. We determined circulating concentrations and ICCs of the inflammatory markers CRP, SAA, and S100A8/9, and the renal function marker CnC in two different cohorts. The plasma concentrations and within-person reproducibility for these protein biomarkers differed between the two cohorts. Plasma levels of SAAt and S100At were higher in WENBIT compared to OMEGA subjects, reflecting prevalent inflammation likely related to established CAD among the WENBIT participants 18,19 . Levels of CnCt were similar in both groups reflecting comparable renal function. ICCs for CRP, SAA and S100A8/9 were highest among the OMEGA subjects generally demonstrating fair-to-good withinperson reproducibility. The lowest ICCs were observed for SAA and S100A8/9 in the WENBIT cohort. Withinperson reproducibility of CnC proteoforms was fair-to-good and similar in both cohorts. Differences in ICCs between proteoforms of the same biomarker were generally small.   20 , improved ICCs for all three inflammatory markers. Although CRP and SAA are both stimulated by IL-6 and highly correlated during inflammation 21 , the ICC improvement was more pronounced for SAA than CRP. This may be related to SAA's role as an acute-phase protein with more pronounced elevation than CRP in response to inflammatory stimuli 22 . Reducing the time interval from 3 to 1 year increased the ICCs of S100A8/9 and CnC, with a concurrent, significant increase in concentrations of both biomarkers over longerterm follow-up. Aging-related elevation of S100At and CnCt levels has recently been associated with chronic inflammation and impaired renal function, respectively 23,24 . Our data suggested that ICC may be impacted by aging related changes such as declining renal function.
Comparison with published data on reproducibility. Others have investigated within-person reproducibility of the selected protein biomarkers, but to our knowledge, this is the first publication to report on proteoform reproducibility. Comparison with other studies is difficult since models for ICC estimations were occasionally not defined and cohorts varied by sample size and time intervals. Several studies evaluating CRP variability, over short (weeks) and long term (≥ 3 y) with sample sizes of dozens to thousands, have reported fairto-good and excellent reproducibility (0.61 < ICC < 0.77) for subjects with CRP values within the normal range (< 10 µg/mL) [25][26][27][28] , which are comparable with the results presented in the present paper. Highest reproducibility was obtained in a short-term study with a 2.5 weeks' time interval 29 . A few studies have determined the ICC for SAAt. A large cohort consisting of 7000 healthy participants determined a value of 0.67 for SAAt, similar to the ICC for the WENBIT group after excluding subjects with CRP > 10 µg/mL 30 . Another study 31 , comparable to OMEGA in size and time interval, investigated 24 healthy participants over 5 weeks and reported a higher ICC of 0.85 for SAAt. Within-person reproducibility of S100At has been reported only once before in a study investigating 207 healthy subjects over a 4-month period 32 . Reproducibility was poor with an ICC of 0.38 but comparable with the value obtained for the WENBIT cohort after 1 year in the present study. The reproducibility of CnCt has been investigated in two small studies, consisting of 10 and 12 healthy individuals 33,34 . In contrast to our study, the reported ICCs were either excellent (ICC = 0.89) or poor (ICC = 0.27), which may be related to the different time spans of 1 and 102 weeks, respectively.
Limitations. Sufficient sample sizes are required for precise estimation of ICCs. The sample sizes of WEN-BIT and OMEGA differed considerably, and 95% CIs varied between 5-26% and 13-86%, respectively. Smaller 95% CIs could have been achieved in OMEGA by repetition of duplicate or triplicate analyses for each time point 35 , but limited sample volume meant only one measurement could be performed. Also, within each cohort 95% CIs of ICCs differed strongly between biomarkers. Relative variation of 95% CIs increases with decreasing ICC, but decrease with increasing sample size. In order to achieve levels of precision for S100At similar to that of CnCt in the WENBIT cohort, sample size would have to be increased to approximately 3000 subjects 36 . A further limitation, includes the analyses of WENBIT and OMEGA samples on different days. Thus, potential effects Distributions of SAA, S100A8/9 and CnC proteoform levels were highly stable over time in both groups. Small significant differences were observed for CnC and S100A8/9 in WENBIT. BL Baseline, END End of follow-up. *, p < 0.05; **, p < 0.01; ***, p < 0.001. www.nature.com/scientificreports/ of preanalytical factors or inter-day variation of the assay on biomarker concentrations cannot be excluded. However, control samples were used in each batch to control for variation. Finally, proportion of male vs female participants in WENBIT was imbalanced (81% vs 19%), but the impact of gender on ICC estimation is expected to be small compared to other factors.

Conclusion
We investigated the within-person reproducibility of the inflammatory biomarkers C-reactive protein (CRP), serum amyloid A (SAA), and calprotectin (S100A8/9), and the renal function marker cystatin C (CnC) and their 16 different proteoforms. Within-person reproducibility was highest in the OMEGA trial with fair-togood reproducibility for all four markers. ICCs of SAA and S100A8/9 in WENBIT appeared to be impacted by the underlying inflammation profile of the cohort. Proteoforms of the same marker demonstrated comparable reproduciblility, and proteoform distributions were highly consistent over time, although ICCs for S100A8/9 and CnC changed with time. This may be linked to a number of factors such as renal function. The presented withinperson reproducibility data will help inform future epidemiological and clinical studies which include these protein biomarkers and allow for correction of potential regression dilution which impacts risk estimations' 1 .